Inhibited Colorectal Cancer Cell Growth Research Articles

MicroRNA-378a-3p prevents initiation and growth of colorectal cancer by fine tuning polyamine synthesis

BackgroundInhibitors of ornithine decarboxylase (ODC) are effective at preventing colorectal cancer (CRC). However, their high toxicity limits their clinical application. This study was aimed to explore the potential of microRNAs (miRNAs) as an inhibitor of ODC.MethodsmiRNA array was used to identify dysregulated miRNAs in CRC tumors of mice and patients. Azoxymethane (AOM)/Dextran Sodium Sulfate (DSS) were used to induce CRC in mice. miRNA function in carcinogenesis was determined by soft-agar colony formation, flow cytometry, and wound healing of CRC cells. Mini-circle was used to deliver miRNA into colons.ResultsMiRNA profiling identified miR-378a-3p (miR-378a) as the most reduced miRNA in CRC tumors of patients and mice treated with AOM/DSS. Pathway array analysis revealed that miR-378a impaired c-MYC and ODC1 pathways. Further studies identified FOXQ1 (forkhead box Q1) and ODC1 as two direct targets of miR-378a. FOXQ1 activated transcription of c-MYC, a transcription activator of ODC1. In addition to directly targeting ODC1, miR-378a also inhibited expression of ODC1 via the FOXQ1-cMYC axis, thereby inhibiting polyamine synthesis in human CRC cells. Phenotypically, by reducing polyamine synthesis, miR-378a induced apoptosis and inhibited proliferation and migration of CRC cells, while disrupting the association of miR-378a with FOXQ1 and ODC1 offset the effects of miR-378a, suggesting that FOXQ1 and ODC1 were required for miR-378a to inhibit CRC cell growth. MiR-378a treatment robustly prevented growth of HCC by inhibiting polyamine synthesis in AOM/DSS mice.ConclusionMiR-378a prevents CRC by inhibiting polyamine synthesis, suggesting its use as a novel ODC inhibitor against CRC.

Toosendanin inhibits colorectal cancer cell growth through the Hedgehog pathway by targeting Shh.

Colorectal cancer (CRC) is one of the most common gastrointestinal cancers worldwide. This complex and often fatal disease has a high mortality rate. The Hedgehog (Hh) signaling pathway is crucial in CRC. Many studies have indicated that Shh is overexpressed in cancer stem cells (CSCs), and shh overexpression is positively correlated with CRC tumorigenesis. New drugs that kill CRC cells through the Hh pathway are needed. Toosendanin (TSN), a natural triterpenoid saponin extracted from the bark or fruit of Melia toosendan Sieb. et Zucc, can inhibit various tumors. Here, we investigated the effects of TSN in CRC and explored the possible targets and mechanisms. Shh-Light Ⅱ cells were treated with TSN and tested by dual luciferase reporter assays to determine the relationship with the Hh pathway. Cell Counting Kit-8 (CCK-8) assays were used to test the inhibitory effects of TSN on CRC cells. The expression of Hh components after TSN treatment was detected using western blots and quantitative reverse transcription polymerase chain reaction. Cellular thermal shift assays confirmed the targets of TSN. The same effects of TSN on xenograft tumor growth were investigated in vivo. The average weight, volume of the finally resected tumor, and the expression of Shh in the TSN-treated groups were significantly lower than those of the control group. This result strongly suggested that TSN administration inhibited CRC growth in vivo. Our research preliminarily demonstrated that the target of TSN was Shh and that TSN inhibits CRC cell growth by inhibiting the Hh pathway, identifying a new anticancer molecular mechanism of TSN in CRC.

SK1 Inhibitor RB005 Induces Apoptosis in Colorectal Cancer Cells through SK1 Inhibition Dependent and Independent Pathway.

Colorectal cancer (CRC) is the fourth leading cause of cancer- related death globally, with a high incidence rate in economically fast-growing countries. Sphingosine- 1-phosphate (S1P) is a bioactive lipid mediator that plays critical roles in cancer cell proliferation, migration, and angiogenesis converted by the isoforms of sphingosine kinase (SK1 and SK2). SK1 is highly expressed in colorectal cancer; its inhibitors suppress the formation of S1P and increase ceramide levels having a pro-apoptotic function. RB005 is a selective SK1 inhibitor and a structural analog of PP2A activator FTY720. The purpose of this study is to test whether RB005, an SK1 inhibitor, can be used as an anticancer agent by inhibiting the growth of colon cancer cells. We performed MTT and colony-forming assay using colon cancer cell lines HT29 and HCT116 cells to examine the cell toxicity effect of RB005. To determine whether apoptosis of RB005 in colon cancer cell line is due to SK1 inhibition or other mechanisms due to its structural similarity with FTY720, we conducted LC/MS, siRNA knockdown, and PP2A activity experiments. RB005 notably inhibited CRC cell growth and proliferation compared to PF543 and ABC294640 by inducing the mitochondria-mediated intrinsic apoptotic pathway. Apoptotic cell death is caused by increased mitochondrial permeability Initiated by the activation of pro-apoptotic protein BAX, increased ceramides, and activation of PP2A. Also, RB005 treatment in HT29 cells did not change the expression level of SK1, but strikingly decreased SK1 activity and S1P levels. All these events of cell death and apoptosis were less effective when SK1 was knocked down by siRNA. This result indicates that RB005 shows the in-vitro anti-CRC effect by inhibiting SK1 activity and PP2A activation, increasing proapoptotic ceramide levels following the activation of the intrinsic apoptotic pathway.

ARHGAP25 expression in colorectal cancer as a biomarker associated with favorable prognosis.

Although progress has been made in the early diagnosis of colorectal cancer (CRC) and in the systemic therapy of patients with CRC, the prognosis for advanced CRC remains poor. Our previous study demonstrated that ARHGAP25 overexpression significantly inhibits CRC cell growth, invasion and migration. However, it was not possible to evaluate and analyze the overall survival (OS) rate of patients with CRC. Thus, the discovery of relevant factors and their expression on the basis of existing research is necessary to predict the OS rate of patients with advanced CRC. Therefore, the aim of the present study was to define the value of Rho GTPase-activating protein 25 (ARHGAP25) expression in predicting the OS rate in patients with CRC. The clinical data of 153 patients with CRC who underwent colorectal resection were retrospectively analyzed. In order to explore the expression of ARHGAP25, immunohistochemical analysis of the tumor tissues of these patients, was performed. Univariate Cox regression analysis was used to assess the prognostic value of ARHGAP25 expression for OS. Multivariate analysis was used to evaluate the effect of ARHGAP25 expression in the presence of other variables. Confounding factors and interaction were assessed by a stratified analysis using ARHGAP25 expression and other variables associated with survival. The univariate analysis revealed that, ARHGAP25 expression was associated with an improved OS in patients with CRC (P<0.05). The multivariate analysis revealed that ARHGAP25 expression was still correlated with an improved OS after adjusting for sex, age, invasion degree, lymph node metastasis, distant metastasis, TNM stage, tumor location, histological type, histological grade, tumor deposits, and postoperative treatment (P<0.05). The stratified analysis demonstrated that the predictive value of ARHGAP25 for the OS of patients with CRC was stronger in males, elderly patients (>70 years old), patients with T3 stage tumor, lymph node metastasis, TNM stage III, right hemicolon location and patients with a poorly differentiated tumor (P<0.05). Overall, our results demonstrated that ARHGAP25 may have an important potential value for improving the prognosis of patients with CRC.

LIM-Domain-Binding Protein 1 Mediates Cell Proliferation and Drug Resistance in Colorectal Cancer.

Objective: It has been shown that LIM-domain-binding protein 1 (LDB1) is involved in the tumorigenesis of several cancers, but its function in colorectal cancer (CRC) has not been fully explained. This study is aimed to investigate whether LDB1 is involved in regulating the cell growth and drug sensitivity of CRC.Methods: To analyze the protein expression of LDB1 in CRC tissues, western blot was used. KM plotter and UALCAN databases were used to predict the prognosis of CRC patients with low or high LDB1 expression. To do the correlation analysis in CRC tissues, GEPIA database was used. CCK-8 assay and xenograft models were used to evaluate the effects of LDB1 in CRC cell growth. An oxaliplatin-resistant cell line was constructed to evaluate the effect of LDB1 in drug sensitivity of CRC cells.Results: Our current research confirmed that LDB1 was upregulated in CRC tumor tissues, and its elevation predicted a poor prognosis for CRC patients. LDB1 was also found positively correlated with CCNA1, BCL2 and BCLW, but negatively correlated with the pro-apoptotic signals (BID, BAX and BAK). Silence of LDB1 significantly inhibited CRC cell growth in vitro, and CRC cells with low expression of LDB1 had a lower tumorigenesis rate in tumor-bearing nude mice. Our experiments also showed that LDB1 silence enhanced the anti-tumor activity of oxaliplatin in CRC cells. The expression of LDB1 was also found increased in oxaliplatin-resistant CRC cell lines, and silence of LDB1 partly restored the antitumor effect of oxaliplatin in an oxaliplatin-resistant CRC cell line.Conclusion: Our current results revealed the roles of LDB1 in the growth and drug resistance of CRC cells, and may provide the new theoretical support for LDB1 as a potential target for the treatment of CRC in the future.

Circ_0007919 exerts an anti-tumor role in colorectal cancer through targeting miR-942–5p/TET1 axis

Circular RNAs (circRNAs) are key regulators in the development of many cancers. The present study was aimed to investigate the mechanism by which circ_0007919 affected colorectal cancer (CRC) progression.The differentially expressed circRNA was screened out by analyzing the expression profile of circRNAs of CRC tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for detecting the expressions of circ_0007919, miR-942–5p, and ten-eleven translocation 1 (TET1) mRNA in CRC tissues and cell lines. Cell growth and migration were assessed by cell counting kit-8 (CCK-8) 5-bromo-2′-deoxyuridine (BrdU) and scratch assays. Bioinformatics analysis and dual-luciferase reporter assay were conducted to predict and validate the targeted relationships between circ_0007919 and miR-942–5p, as well as between miR-942–5p and TET1 mRNA. Besides, Western blot was conducted for detecting TET1 protein expression in CRC cells. It was revealed that, in CRC tissues and cell lines, circ_0007919 and TET1 expressions were reduced whereas miR-942–5p expression was enhanced. It was also revealed that circ_0007919 overexpression markedly suppressed CRC cell growth and migration. In addition, circ_0007919 could competitively bind with miR-942–5p to increase the expression of miR-942–5p’s target gene TET1. Collectively, circ_0007919 inhibits CRC cell growth and migration via regulating the miR-942–5p/TET1 axis. This study helps to better understand the molecular mechanism of CRC progression.

Combination gut microbiota modulation and chemotherapy for orthotopic colorectal cancer therapy

Gut microbiota is closely related to the tumorigenesis and progression of colorectal cancer (CRC), especially Fusobacterium nucleatum (Fn) could promote colorectal tumor growth and cause chemoresistance. Here, we developed a tumor-triggered release drug delivery gel to inhibit CRC cell growth in situ by intestinal perfusion. Antibiotic metronidazole (MTZ) and 5-fluorouracil (5-FU), the first-line chemotherapy drug of CRC, are individually incorporated into metal polyphenol network-coated mesoporous silica nanoparticle (MSN), and then blended with carboxymethyl cellulose (CMC) to obtain an anti-CRC gel, AB-Gel. In vivo studies showed that AB-Gel could efficiently inhibit the growth and metastasis of CRC in an orthotopic mouse model attributing to the MTZ-induced modulation in the CRC-related microbiota. Chemotherapy efficacy against orthotopic CRC was significantly enhanced by the elimination of Fn. This strategy based on the combination of gut microbiota modulation and chemotherapy would be considered as an enhanced strategy for CRC treatment with potential use in the clinic.

Expression Profile and Prognostic Value of Wnt Signaling Pathway Molecules in Colorectal Cancer.

Colorectal cancer (CRC) is a heterogeneous disease with changes in the genetic and epigenetic levels of various genes. The molecular assessment of CRC is gaining increasing attention, and furthermore, there is an increase in biomarker use for disease prognostication. Therefore, the identification of different gene biomarkers through messenger RNA (mRNA) abundance levels may be useful for capturing the complex effects of CRC. In this study, we demonstrate that the high mRNA levels of 10 upregulated genes (DPEP1, KRT80, FABP6, NKD2, FOXQ1, CEMIP, ETV4, TESC, FUT1, and GAS2) are observed in CRC cell lines and public CRC datasets. Moreover, we find that a high mRNA expression of DPEP1, NKD2, CEMIP, ETV4, TESC, or FUT1 is significantly correlated with a worse prognosis in CRC patients. Further investigation reveals that CTNNB1 is the key factor in the interaction of the canonical Wnt signaling pathway with 10 upregulated CRC-associated genes. In particular, we identify NKD2, FOXQ1, and CEMIP as three CTNNB1-regulated genes. Moreover, individual inhibition of the expression of three CTNNB1-regulated genes can cause the growth inhibition of CRC cells. This study reveals efficient biomarkers for the prognosis of CRC and provides a new molecular interaction network for CRC.

Two naturally derived small molecules disrupt the sineoculis homeobox homolog 1-eyes absent homolog 1 (SIX1-EYA1) interaction to inhibit colorectal cancer cell growth.

Background:Emerging evidence indicates that the sineoculis homeobox homolog 1−eyes absent homolog 1 (SIX1–EYA1) transcriptional complex significantly contributes to the pathogenesis of multiple cancers by mediating the expression of genes involved in different biological processes, such as cell-cycle progression and metastasis. However, the roles of the SIX1–EYA1 transcriptional complex and its targets in colorectal cancer (CRC) are still being investigated. This study aimed to investigate the roles of SIX1–EYA1 in the pathogenesis of CRC, to screen inhibitors disrupting the SIX1–EYA1 interaction and to evaluate the efficiency of small molecules in the inhibition of CRC cell growth.Methods:Real-time quantitative polymerase chain reaction and western blotting were performed to examine gene and protein levels in CRC cells and clinical tissues (collected from CRC patients who underwent surgery in the Department of Integrated Traditional and Western Medicine, West China Hospital of Sichuan University, between 2016 and 2018, n = 24). In vivo immunoprecipitation and in vitro pulldown assays were carried out to determine SIX1–EYA1 interaction. Cell proliferation, cell survival, and cell invasion were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, clonogenic assay, and Boyden chamber assay, respectively. The Amplified Luminescent Proximity Homogeneous Assay Screen (AlphaScreen) method was used to obtain small molecules that specifically disrupted SIX1–EYA1 interaction. CRC cells harboring different levels of SIX1/EYA1 were injected into nude mice to establish tumor xenografts, and small molecules were also injected into mice to evaluate their efficiency to inhibit tumor growth.Results:Both SIX1 and EYA1 were overexpressed in CRC cancerous tissues (for SIX1, 7.47 ± 3.54 vs.1.88 ± 0.35, t = 4.92, P = 0.008; for EYA1, 7.61 ± 2.03 vs. 2.22 ± 0.45, t = 6.73, P = 0.005). The SIX1/EYA1 complex could mediate the expression of two important genes including cyclin A1 (CCNA1) and transforming growth factor beta 1 (TGFB1) by binding to the myocyte enhancer factor 3 consensus. Knockdown of both SIX1 and EYA1 could decrease cell proliferation, cell invasion, tumor growth, and in vivo tumor growth (all P < 0.01). Two small molecules, NSC0191 and NSC0933, were obtained using AlphaScreen and they could significantly inhibit the SIX1–EYA1 interaction with a half-maximal inhibitory concentration (IC50) of 12.60 ± 1.15 μmol/L and 83.43 ± 7.24 μmol/L, respectively. Administration of these two compounds could significantly repress the expression of CCNA1 and TGFB1 and inhibit the growth of CRC cells in vitro and in vivo.Conclusions:Overexpression of the SIX1/EYA1 complex transactivated the expression of CCNA1 and TGFB1, causing the pathogenesis of CRC. Pharmacological inhibition of the SIX1–EYA1 interaction with NSC0191 and NSC0933 significantly inhibited CRC cell growth by affecting cell-cycle progression and metastasis.

CircRNA_103948 inhibits autophagy in colorectal cancer in a ceRNA manner.

Circular RNA (circRNA) is implicated in many types of cancer; however, the expression and role of circRNAs in colorectal cancer (CRC) remains poorly understood. In this study, a circRNA microarray assay was performed to detect abnormally expressed circRNAs in CRC, and tissue arrays were used to determine the prognosis for CRC patients. Cell counting kit-8, clone formation, wound healing, and transwell assays were used to evaluate cell functions in vitro, and a mouse subcutaneous tumor model was designed for in vivo analysis. Autophagy was observed using confocal laser scanning and transmission electron microscopy. The expression of circRNA, miRNA, and mRNA was detected using qPCR; western blot, RNA pull-down assay, RNA immunoprecipitation, and dual luciferase assessment were applied for mechanistic studies. We found that circRNA_103948 expression is upregulated in CRC tissues, compared with adjacent normal tissues, and associated with poor prognosis. Knockdown of circRNA_103948 suppressed CRC both in vitro and in vivo. Mechanistically, circRNA_103948 could directly bind to miR-1236-3p and relieve suppression of the target TPT1. Furthermore, circRNA_103948 inhibited autophagy of CRC cells. Taken together, circRNA_103948 knockdown inhibited CRC cell growth by targeting miR-1236-3p/TPT1 axis-mediated autophagy. Thus, the circRNA_103948/miR-1236-3p/TPT1 axis affects CRC progression via modulation of autophagy.

Wild-type IDH2 protects nuclear DNA from oxidative damage and is a potential therapeutic target in colorectal cancer.

Although the role of isocitrate dehydrogenase (IDH) mutation in promoting cancer development has been well-characterized, the impact of wild-type IDH on cancer cells remains unclear. Here we show that the wild-type isocitrate dehydrogenase 2 (IDH2) is highly expressed in colorectal cancer (CRC) cells, and plays an unexpected role in protecting the cancer cells from oxidative damage. Genetic abrogation of IDH2 in CRC cells leads to reactive oxygen species (ROS)-mediated DNA damage and an accumulation of 8-oxoguanine with DNA strand breaks, which activates DNA damage response (DDR) with elevated γH2AX and phosphorylation of ataxia telangiectasia-mutated (ATM) protein, leading to a partial cell cycle arrest and eventually cell senescence. Mechanistically, the suppression of IDH2 results in a reduction of the tricarboxylic acid (TCA) cycle activity due to a decrease in the conversion of isocitrate to α-ketoglutarate (α-KG) with a concurrent decrease in NADPH production, leading to ROS accumulation and oxidative DNA damage. Importantly, abrogation of IDH2 inhibits CRC cell growth in vitro and in vivo, and renders CRC cells more vulnerable to DNA-damaging drugs. Screening of an FDA-approved drug library has identified oxaliplatin as a compound highly effective against CRC cells when IDH2 was suppressed. Our study has uncovered an important role of the wild-type IDH2 in protecting DNA from oxidative damage, and provides a novel biochemical basis for developing metabolic intervention strategy for cancer treatment.

DAB2IP decreases cell growth and migration and increases sensitivity to chemotherapeutic drugs in colorectal cancer.

BackgroundColorectal cancer (CRC) is one of the most common cancers worldwide with high rates of invasiveness and mortality. DAB2IP (DOC2/DAB2 interactive protein) is a member of the RAS-GTPase-activating protein (RAS-GAP) family that shows a suppressive effect on cancer progression, is downregulated in several cancers. However, the role of DAB2IP in CRC remains elusive.MethodsExpression of DAB2IP was evaluated in human CRC tissues using immunohistochemistry (IHC), quantitative real-time reverse transcription PCR (qRT-PCR) and immunoblotting. Knockdown and overexpression of DAB2IP in CRC cells were achieved by transfecting siRNAs and DAB2IP expression vectors and assessed by qRT-PCR and immunoblotting. CCK-8, colony formation, wound-healing, and transwell assays were used to evaluate CRC cell growth, migration, and sensitivity to chemotherapeutic drugs. The cell cycle was analyzed by propidium iodide (PI) staining and flow cytometry. Cell apoptosis was evaluated by Annexin V-DAPI double staining and flow cytometry. The effect of DAB2IP overexpression on tumor formation was explored by an in vivo tumorigenesis assay. Finally, immunoblotting was performed to examine the molecules related to the action of DAB2IP in CRC.ResultsCompared with para-cancer tissues, there was a marked decrease of DAB2IP expression in surgically excised CRCs. In cultured CRC cells, enforced expression of DAB2IP inhibited cell growth and migration and sensitized the cells to DNA-acting cisplatin, oxaliplatin, and doxorubicin but not 5-fluorouracil (5-FU). In contrast, knockdown of DAB2IP produced the opposite effect. Moreover, DAB2IP overexpression hindered tumor growth in vivo. We further found that DAB2IP regulated the expression of cell growth, epithelial-mesenchymal transition (EMT), and apoptosis-related proteins in CRC cells and inhibited the phosphorylation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK).ConclusionsExpression of DAB2IP inhibited CRC cell growth and migration and sensitized CRC cells to chemotherapeutic drugs. Inhibition of the phosphorylation of AKT and ERK is associated with the effects of DAB2IP expression. Restoration of DAB2IP expression may be a novel target for treating CRC.

Squalene epoxidase promotes colorectal cancer cell proliferation through accumulating calcitriol and activating CYP24A1-mediated MAPK signaling.

BackgroundColorectal cancer (CRC) is one of the most malignant tumors with high incidence, yet its molecular mechanism is not fully understood, hindering the development of targeted therapy. Metabolic abnormalities are a hallmark of cancer. Targeting dysregulated metabolic features has become an important direction for modern anticancer therapy. In this study, we aimed to identify a new metabolic enzyme that promotes proliferation of CRC and to examine the related molecular mechanisms.MethodsWe performed RNA sequencing and tissue microarray analyses of human CRC samples to identify new genes involved in CRC. Squalene epoxidase (SQLE) was identified to be highly upregulated in CRC patients. The regulatory function of SQLE in CRC progression and the therapeutic effect of SQLE inhibitors were determined by measuring CRC cell viability, colony and organoid formation, intracellular cholesterol concentration and xenograft tumor growth. The molecular mechanism of SQLE function was explored by combining transcriptome and untargeted metabolomics analysis. Western blotting and real‐time PCR were used to assess MAPK signaling activation by SQLE.ResultsSQLE‐related control of cholesterol biosynthesis was highly upregulated in CRC patients and associated with poor prognosis. SQLE promoted CRC growth in vitro and in vivo. Inhibition of SQLE reduced the levels of calcitriol (active form of vitamin D3) and CYP24A1, followed by an increase in intracellular Ca2+ concentration. Subsequently, MAPK signaling was suppressed, resulting in the inhibition of CRC cell growth. Consistently, terbinafine, an SQLE inhibitor, suppressed CRC cell proliferation and organoid and xenograft tumor growth.ConclusionsOur findings demonstrate that SQLE promotes CRC through the accumulation of calcitriol and stimulation of CYP24A1‐mediated MAPK signaling, highlighting SQLE as a potential therapeutic target for CRC treatment.

Rhein Suppresses Colorectal Cancer Cell Growth by Inhibiting the mTOR Pathway In Vitro and In Vivo.

Simple SummaryColorectal cancer (CRC) is the fourth most common cancer and the second most common cause of cancer-related deaths globally. Rhein is a natural anthraquinone extract from rhubarb, which exhibits potent anticancer activity in various cancers. In this study, we show that rhein significantly inhibited the growth, migration, and invasion of CRC cells by directly binding to mTOR and inhibiting the mTOR signaling pathway. Rhein promotes mTOR degradation through the ubiquitin–proteasome pathway. In addition, rhein significantly suppressed tumor growth in a xenograft mouse model without obvious toxicity. Our results indicate that rhein is a promising anticancer agent that may be useful for the prevention and treatment of CRC.Colorectal cancer (CRC) is one of the leading causes of mortality and morbidity in the world. Rhein has demonstrated therapeutic effects in various cancer models. However, its effects and underlying mechanisms of action in CRC remain poorly understood. We investigated the potential anticancer activity and underlying mechanisms of rhein in CRC in vitro and in vivo. Cell viability and anchorage-independent colony formation assays were performed to examine the antigrowth effects of rhein on CRC cells. Wound-healing and Transwell assays were conducted to assess cell migration and invasion capacity. Cell cycle and apoptosis were investigated by flow cytometry and verified by immunoblotting. A tissue microarray was used to detect mTOR expression in CRC patient tissues. Gene overexpression and knockdown were done to analyze the function of mTOR in CRC. The anticancer effect of rhein in vivo was assessed in a CRC xenograft mouse model. The results show that rhein significantly inhibited CRC cell growth by inducing S-phase cell cycle arrest and apoptosis. Rhein inhibited CRC cell migration and invasion through the epithelial–mesenchymal transition (EMT) process. mTOR was highly expressed in CRC cancer tissues and cells. Overexpression of mTOR promoted cell growth, migration, and invasion, whereas mTOR knockdown diminished these phenomena in CRC cells in vitro. In addition, rhein directly targeted mTOR and inhibited the mTOR signaling pathway in CRC cells. Rhein promoted mTOR degradation through the ubiquitin-proteasome pathway. Intraperitoneal administration of rhein inhibited HCT116 xenograft tumor growth through the mTOR pathway. In conclusion, rhein exerts anticancer activity in vitro and in vivo by targeting mTOR and inhibiting the mTOR signaling pathway in CRC. Our results indicate that rhein is a potent anticancer agent that may be useful for the prevention and treatment of CRC.

Fangchinoline exerts anticancer effects on colorectal cancer by inducing autophagy via regulation AMPK/mTOR/ULK1 pathway

Autophagy has become a promising target for cancer therapy. Fangchinoline (Fan) has been shown to exert anticancer effects in some types of cancers. However, the anticancer effects on colorectal cancer (CRC) and the underlying mechanisms have never been elucidated. More specifically, regulation of autophagy in CRC by Fan has never been reported before. In the present study, Fan was found to induce apoptosis and autophagic flux in the CRC cell lines HT29 and HCT116, which was reflected by the enhanced levels of LC3-II protein and p62 degradation, and the increased formation of autophagosomes and puncta formation by LC3-II. Meanwhile, combination with the early-stage autophagy inhibitor 3-methyladenine (3-MA) but not the late-stage autophagy inhibitor chloroquine (CQ) further increased Fan-induced cell death, which suggested the cytoprotective function of autophagy induced by Fan in both HT29 and HCT116 cells. Moreover, Fan treatment demonstrated a dose- and time-dependently increase in the phosphorylation of AMPK and decrease in the phosphorylation of mammalian target of rapamycin (mTOR) and ULK1, leading to the activation of the AMPK/mTOR/ULK1 signaling pathway. Furthermore, in the HT29 xenograft model, Fan inhibited tumor growth in vivo. These results indicate that Fan inhibited CRC cell growth both in vitro and in vivo and revealed a new molecular mechanism involved in the anticancer effect of Fan on CRC, suggesting that Fan is a potent autophagy inducer and might be a promising anticancer agent.

LINC00667/miR-449b-5p/YY1 axis promotes cell proliferation and migration in colorectal cancer

BackgroundLong non-coding RNAs (lncRNAs) have been defined as vital regulators in the progression of human cancers, including colorectal cancer (CRC). Long intergenic non-protein coding RNA 667 (LINC00667) is a tumor promoter in several cancer types, while its role in CRC remains to be unmasked. This study focused on exploring the potential function and regulatory mechanism of LINC00667 in CRC.MethodsqRT-PCR analysis was applied to detect the expression of LINC00667 in CRC cells. Loss-of function assays revealed the role of LINC00667 silencing in regulating CRC cell proliferation, apoptosis and migration. In vivo study demonstrated the effect of LINC00667 silencing on CRC cell growth. Mechanism experiments were conducted to determine the upstream or the downstream molecular mechanism of LINC00667 in CRC cells.ResultsLINC00667 was expressed at high level in CRC cells. LINC00667 knockdown significantly inhibited CRC cell growth and migration. YY1 transcription factor induced the upregulation of LINC00667 in CRC cells by transcriptionally activating LINC00667. In addition, miR-449b-5p could interact with LINC00667 in CRC cells. Intriguingly, miR-449b-5p directly targeted to YY1, thus inhibiting YY1 expression. YY1 recovered the CRC cell functions impaired by LINC00667 silencing.ConclusionsLINC00667 is transcriptionally activated by YY1 and promotes cell proliferation and migration in CRC by sponging miR-449b-5p to upregulate YY1.

MicroRNA-1307-3p accelerates the progression of colorectal cancer via regulation of TUSC5.

The aim of the present study was to explore the roles of microRNA-1307-3p (miR-1307-3p) in colorectal cancer (CRC). Firstly, the expression level of miR-1307-3p in CRC cells was measured using reverse transcription-quantitative PCR. Subsequently, Cell Counting Kit-8 and Transwell invasion assays were performed to evaluate the effects of miR-1307-3p on CRC cell proliferation and invasion, respectively. Bioinformatics tools and dual luciferase reporter assays were used to validate the targets of miR-1307-3p. Rescue experiments were performed to confirm tumor suppressor candidate 5 (TUSC5) as a functional target of miR-1307-3p. miR-1307-3p levels were revealed to be upregulated in CRC cells when compared with the normal human epithelial cell line. Knockdown of miR-1307-3p inhibited CRC cell growth and invasiveness. Bioinformatics analysis and dual-luciferase activity reporter assays demonstrated that miR-1307-3p binds the 3'-untranslated region of TUSC5. Finally, rescue experiments validated that miR-1307-3p was able to regulate CRC cell behaviors via regulating TUSC5 expression. Together, the current results indicate that miR-1307-3p functions as an oncogenic miRNA via targeting TUSC5 in CRC.

CircFAT1 Suppresses Colorectal Cancer Development Through Regulating miR-520b/UHRF1 Axis or miR-302c-3p/UHRF1 Axis.

Background: It was reported that circular RNAs (circRNAs) exerted important functions in various human cancers. However, the function of circFAT1 was less known. The purpose of this study was to reveal the functional mechanism of circFAT1 in colorectal cancer (CRC). Materials and Methods: Quantitative real-time polymerase chain reaction and Western blot assay were used to detect the levels of genes. Cell proliferation ability was assessed by 3-(4, 5-dimethyl-2-thiazoyl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Flow cytometry was used to investigate cell apoptosis rate. The glucose consumption and lactate production were determined using related kits. Furthermore, the interaction between circFAT1 or ubiquitin-like PHD and RING finger domain-containing protein 1 (UHRF1) and miR-520b or miR-302c-3p was predicted by starbase3.0, and then confirmed by the dual-luciferase reporter assay. Besides, xenograft experiment was performed to analyze the effect of circFAT1 on tumor growth in vivo. Results: The levels of circFAT1 and UHRF1 were increased, as well as the levels of miR-520b and miR-302c-3p were decreased in CRC tissues and cells. CircFAT1 knockdown suppressed cell proliferation, cycle, and glycolysis as well as induced apoptosis. Interestingly, circFAT1 was a sponge of miR-520b and miR-302c-3p, and miR-520b and miR-302c-3p could target UHRF1. Both miR-520b overexpression and miR-302c-3p overexpression inhibited CRC cell growth. Furthermore, both miR-520b knockdown and miR-302c-3p depletion weakened the effect of circFAT1 knockdown on the growth of CRC cells. Besides, circFAT1 depletion repressed tumor growth in vivo. Conclusion: The authors' findings suggested that circFAT1 upregulated UHRF1 to affect CRC cell proliferation, apoptosis, and glycolysis through targeting miR-520b and miR-302c-3p, providing theoretical basis for the treatment of CRC.

The inhibitory effect of ECG and EGCG dimeric procyanidins on colorectal cancer cells growth is associated with their actions at lipid rafts and the inhibition of the epidermal growth factor receptor signaling

Colorectal cancer (CRC) is one of the most common cancers worldwide. Epidemiological studies indicate that consumption of fruits and vegetables containing procyanidins is associated with lower CRC risk. This study investigated the capacity of two dimeric procyanidins composed of epicatechin gallate (ECG) or epigallocatechin gallate (EGCG) isolated from persimmons, to inhibit CRC cell growth and promote apoptosis, characterizing the underlying mechanisms. ECG and EGCG dimers reduced the growth of five human CRC cell lines in a concentration (10–60 μM)- and time (24–72 h)-dependent manner, with a 72 h-IC50 value in Caco-2 cells of 10 and 30 μM, respectively. ECG and EGCG dimers inhibited Caco-2 cell proliferation by arresting the cell cycle in G2/M phase and by inducing apoptosis via the mitochondrial pathway. In addition, ECG and EGCG dimers inhibited cell migration, invasion, and adhesion, decreasing the activity of matrix metalloproteinases (MMP-2/9). Mechanistically, ECG and EGCG dimers inhibited the activation of lipid raft-associated epidermal growth factor (EGF) receptor (EGFR), without affecting its localization at lipid rafts. In particular, ECG and EGCG dimers reduced EGFR phosphorylation at Tyr1068 residue, prevented EGFR dimerization and activation upon stimulation, and induced EGFR internalization both in the absence and presence of EGF. Furthermore, ECG and EGCG dimers increased EGFR phosphorylation at Tyr1045 residue, providing a docking site for ubiquitin ligase c-Cbl and induced EGFR degradation by the proteasome. Downstream of EGFR, ECG and EGCG dimers inhibited the activation of the MEK/ERK1/2 and PI3K/AKT signaling pathways, downregulating proteins involved in the modulation of cell survival. In conclusion, ECG and EGCG dimers reduced CRC cell growth by inhibiting EGFR activation at multiple steps, including the disruption of lipid rafts integrity and promoting EGFR degradation. These results shed light on a potential molecular mechanism on how procyanidins-rich diets may lower CRC risk.

SEZ6L2 knockdown impairs tumour growth by promoting caspase-dependent apoptosis in colorectal cancer.

Seizure‐related 6 homolog (mouse)‐like 2 (SEZ6L2) was shown to be involved in transcription of a type 1 transmembrane protein for regulating cell fate. Until now, the expression and function of SEZ6L2 in various cancers, including colorectal cancer (CRC), were unclear. In the present study, we determined the expression of SEZ6L2 in a tissue microarray from patients with CRC and then, analysed the correlation between SEZ6L2 expression and the prognosis of the patients. Furthermore, the potential function of SEZ6L2 in CRC was determined using cell counting kit, colony formation assay and xenograft model in vitro and in vivo. Flow cytometry, Western blotting, immunohistochemical staining and a blocking experiment were employed to investigate the underlying mechanism of SEZ6L2 regulating CRC growth. Our results indicated that SEZ6L2 was significantly up‐regulated in tumour tissues of patients with CRC compared with adjacent normal tissues. Up‐regulation of SEZ6L2 was correlated with a poor prognosis in patients with CRC. In vitro experiments suggested that the knockdown of SEZ6L2 inhibits CRC cell growth and colony formation, but it has no significant impact on the invasion. The antitumour effects of shSEZ6L2 were also confirmed by a xenograft model. Investigations of the mechanisms indicated that the knockdown of SEZ6L2 impairs the growth of the CRC cells by inducing caspase‐dependent apoptosis, which was mediated by mitochondria‐related proteins. Furthermore, SEZ6L2 expression was inversely correlated with the expression of cytochrome C in malignant tissues in patients with CRC. Collectively, the present study indicates that SEZ6L2 is a potential prognosis biomarker and therapy target for CRC.